Compound

ABSTRACT

The present invention relates inter alia to a compound of formula (I) 
     
       
         
         
             
             
         
       
     
     and to compositions comprising the same and to the use of the compounds and to compositions of the compounds in treatment, for example in the treatment of inflammatory diseases, in particular respiratory inflammatory disease. The invention also extends to methods of making the said compounds.

FIELD OF THE INVENTION

The invention relates to compounds that inhibit phosphoinositide3-kinases, (PI3 kinases, PI3K). In particular the disclosure relates tocompounds that inhibit the PI3K delta sub-type and, in addition, thegamma sub-type thereof, and to their use in therapy, including inpharmaceutical combinations, especially in the treatment of inflammatorydiseases, including inflammatory diseases of the lung, such as COPD andasthma. The invention also extends to methods of preparing the saidcompounds and to pharmaceutical compositions comprising the same.

BACKGROUND OF THE INVENTION

Lipid kinases catalyse the phosphorylation of lipids to produce speciesinvolved in the regulation of a wide range of physiological processes,including cellular migration and adhesion. The PI3 kinases (PI3K) aremembrane associated proteins and belong to the class of enzymes whichcatalyse the phosphorylation of lipids which are themselves associatedwith cell membranes. The PI3K delta isozyme (PI3K 6) is one of fourisoforms of type I PI3K kinases responsible for generating various3′-phosphorylated phosphoinositides, that mediate cellular signallingand have been implicated in inflammation, growth factor signalling,malignant transformation and immunity [See Review by Rameh, L. E. andCantley, L. C. J. Biol. Chem., 1999, 274:8347-8350.].

The involvement of PI3K in controlling inflammation has been confirmedin several models using pan-active PI3K inhibitors, such as LY-294002and Wortmannin [Ito, K. et al., J Pharmacol. Exp. Ther., 2007,321:1-8.]. Recent studies have been conducted using either selectivePI3K inhibitors or in knock-out mice lacking a specific enzyme isoformas described below. These studies have demonstrated the role of pathwayscontrolled by PI3K enzymes in inflammation. The PI3K δ selectiveinhibitor IC-87114 was found to inhibit airways hyper-responsiveness,IgE release, pro-inflammatory cytokine expression, inflammatory cellaccumulation into the lung and vascular permeability inovalbumin-sensitized, ovalbumin-challenged mice [Lee, K. S. et al., J.Allergy Clin. Immunol., 2006, 118:403-409 and Lee, K. S. et al., FASEBJ., 2006, 20:455-65.]. In addition, IC-87114 lowered neutrophilaccumulation in the lungs of mice and neutrophil function, stimulated byTNFα [Sadhu, C. et al., Biochem. Biophys. Res. Commun., 2003,308:764-9].

The PI3K δ isoform is activated by insulin and other growth factors, aswell as by G-protein coupled protein signaling and inflammatorycytokines. Furthermore, studies using knockout mice revealed thatactivation of PI3K γ may be important in the pathogenesis of asthma. Forexample, murine mast cell responses are exacerbated in vitro and in vivoby autocrine signals which require functional PI3K γ. Mice that lackedPI3K γ did not exhibit edema when challenged by passive systemicanaphylaxis [Wymann M. P. et al., Biochem. Soc. Trans., 2003,31:275-80.]. Thus PI3K γ relays inflammatory signals through variousG-protein coupled receptors (GPCRs), especially by controlling mast cellfunction. Eosinophil accumulation in ovalbumin sensitised and challengedmice was also reported to be inhibited in these PI3K γ-deficient mice,as compared with wild-type animals [Lim D. H. et al., Am. J. Physiol.Lung Cell. Mol. Physiol., 2009, 296(2):L210-L219]. Finally, treatmentwith a PI3K γ inhibitor attenuated IL-13-augmented airway contractilityof lung slices [Jiang H. et al., J. Pharmacol. Exp. Ther., 2012,342(2):305-11.].

Recently the PI3K dual δ/γ inhibitor TG100-115 was reported to inhibitpulmonary eosinophilia and decrease interleukin-13 levels, mucinaccumulation and airways hyper-responsiveness in a murine model, whenadministered by aerosolisation. The same authors also reported that thecompound was able to inhibit pulmonary neutrophilia elicited by eitherLPS or cigarette smoke [Doukas, J. et al., J. Pharmacol. Exp. Ther.,2009, 328:758-765.]. Other small molecule inhibitors of PI3K δ and γwere reported to produce superior inhibition of LPS induced TNFαproduction and T cell activation when compared with PI3K δ selectiveinhibitors [Williams O. et al., Chem Biol., 2010, 17(2):123-34.].

Since it is also activated by oxidative stress, the PI3K δ isoform islikely to be relevant as a target for therapeutic intervention in thosediseases where a high level of oxidative stress is implicated.Downstream mediators of the PI3K signal transduction pathway include Akt(a serine/threonine protein kinase) and the mammalian target ofrapamycin, the enzyme mTOR. Recent work has suggested that activation ofPI3K δ, leading to phosphorylation of Akt, is able to induce a state ofcorticosteroid resistance in otherwise corticosteroid-sensitive cells[To, Y. et al., Am. J. Respir. Crit. Care Med., 2010, 182:897-904.].These observations have led to the hypothesis that this signallingcascade could be one mechanism responsible for thecorticosteroid-insensitivity of inflammation observed in the lungs ofpatients suffering from COPD, as well as in those asthmatics who smoke,thereby subjecting their lungs to increased oxidative stress. Indeed,theophylline, a compound used in the treatment of both COPD and asthma,has been suggested to reverse steroid insensitivity through mechanismsinvolving interaction with pathways controlled by PI3 kinase δ [To, Y.et al., Am. J. Respir. Crit. Care Med., 2010, 182:897-904.].

At present the mainstay of treatment for both asthma and COPD is inhaledtherapy, using a combination of corticosteroids, muscarinic antagonistsand β₂-agonists, as judged clinically appropriate. One way of addressingthe unmet medical needs in COPD and asthma is to identify new inhaledmedicines, which have the potential to provide significant benefit whenused either as a monotherapy or in combination with one or moremedicaments from these three pharmacological classes. Therefore, thereremains a need to identify and develop isoform selective PI3K inhibitorswhich have the potential to provide enhanced therapeutic efficacy inasthma, COPD and other inflammatory diseases.

WO 2012/052753 discloses:6-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamide, referred to herein as prior art Compound A.

WO 2011/048111 discloses certain3-benzyl-5-alkynyl-quinazolin-4(3H)-ones

A compound example disclosed therein is2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-fluorobenzyl)-5-(3-(2-(2-methoxyethoxy)ethoxy)prop-1-ynyl)quinazolin-4(3H)-one, referred to herein as Example 50.

Neither of these prior art compounds possess the same advantageousprofile of the compound of formula (I) described herein.

SUMMARY OF THE INVENTION

According to the invention, there is provided a compound of formula (I):

or a pharmaceutically acceptable salt thereof, including allstereoisomers, tautomers and isotopic derivatives thereof.

A comparison of the in vitro profiles of prior art compounds Compound Aand Example 50 with the compound of the present disclosure is presentedherein below (see Table 1 in Experimental Section). The compound of thepresent invention is a particularly potent dual PI3K δ/γ isoforminhibitor, a pharmacological feature that confers upon it a distinct andadvantageous therapeutic profile over compounds disclosed previously.This aspect of the invention is particularly evident from the in vivoprofile of the compound of formula (I) in animal models that arepredictors of therapeutic efficacy and clinical benefit in pulmonaryinflammation (see Tables 4-8 in the Experimental Section).

In one embodiment the compound of the present disclosure has improvedinhibitory activity over prior art compounds when compared in an in vivoassay that measures the ability of a test substance to inhibit poly ICinduced influx of neutrophils into mouse lung. The improvementdemonstrated may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 fold (or more) overknown compounds, previously disclosed in the art.

Activity in the in vivo poly IC assay is considered indicative of thepotential of the compound of the present invention to inhibitvirus-induced exacerbations in patients suffering with asthma or withCOPD. It is thought that viral infections induce disease exacerbationsby worsening inflammation in the lungs of patients and by generating asteroid resistant phenotype. Poly IC stimulates one of the mechanismsthrough which viruses are pro-inflammatory.

The drug-like properties of the compound described herein, including itsintrinsic physical and chemical stability, solubility profile, andespecially its distinctive biological activity render the said compoundparticularly suitable for use as a pharmaceutical agent and inparticular for the treatment of inflammatory mediated diseases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 a: is a bar graph representing the effects of treatment withcompound (I) or compound A on poly-I:C-induced neutrophil accumulationin mouse airways.

FIG. 1 b: shows a comparison of the inhibitory potencies of compound (I)against compound A on poly-I:C-induced neutrophil accumulation in mouseairways

FIG. 2 a: is a bar graph representing the effect of treatment withcompound (I) or compound A on cigarette smoke-induced macrophageaccumulation in murine BALF.

FIG. 2 b: is a bar graph representing the effect of treatment withcompound (I) or compound A on cigarette smoke-induced neutrophilaccumulation in murine BALF.

DETAILED DESCRIPTION OF THE INVENTION

The term inhibitor as employed herein is intended to refer to a compoundthat reduces (for example by at least 10%, 20%, 30%, 40%, 50% or more)or eliminates the biological activity of the target protein, for examplethe PI3K δ isozyme, in an in vitro enzyme assay.

The term delta/gamma (δ/γ) inhibitor as employed herein is intended torefer to the fact that the compound inhibits, to some degree, inhibitoryactivity at both enzyme isoforms although not necessarily to the sameextent.

The compound of the present disclosure is active in cell based screeningsystems and thereby demonstrates that it possesses suitable propertiesfor penetrating cells and is able to exert intracellular pharmacologicaleffects (see Table 2 and Table 3 in the Experimental Section).

The compound of the present disclosure has therapeutically relevant anddesirable pharmaceutical properties for a medicament, for examplephysico-chemical features including adequate chemical andphotostability, an appropriate solubility profile and potent activity.

In one embodiment there is a provided a pharmaceutically acceptable acidaddition salt of the compound of the invention.

The pharmaceutically acceptable acid addition salts referred tohereinabove are meant to comprise the therapeutically active, non-toxic,acid addition salts that the compounds of formula (I) are able to form.These pharmaceutically acceptable acid addition salts can convenientlybe obtained by treating the free base form of the compound of formula(I) with an example of such appropriate acids. Appropriate acidscomprise, for example, inorganic acids such as hydrochloric acid,hydrobromic acid, and sulfuric, and phosphoric acids and the like; ororganic acids such as, for example, acetic, propanoic, hydroxyacetic,lactic, malonic, succinic, maleic, fumaric, malic, tartaric, citric,methanesulfonic, para-toluenesulfonic, cyclamic, salicylic,para-aminosalicylic, pamoic acid and the like.

Examples of salts of the compound of formula (I) include allpharmaceutically acceptable salts, such as, without limitation, acidaddition salts of mineral acids such as HCl and HBr salts and additionsalts of organic acids such as a methanesulfonic acid salt. Furtherexamples include sulphuric acid salts and phosphoric acid salts.

In one embodiment there is provided2-((4-amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-(3-(2-(2-methoxyethoxy)ethoxy)prop-1-yn-1-yl)-3-(2-(trifluoromethyl)benzyl)quinazolin-4(3H)-oneas the free base.

The invention also extends to solvates of the compounds disclosedherein. Examples of solvates include hydrates.

The compounds of the disclosure include those where the atom specifiedis a naturally occurring or non-naturally occurring isotope. In oneembodiment the isotope is a stable isotope. Thus the compounds of thedisclosure include, for example those containing one or more deuteriumatoms in place of hydrogen atoms and the like.

The disclosure also extends to all polymorphic forms of the compoundsherein defined.

The compounds of formula (I) may be conveniently prepared by a processcomprising reacting a compound of formula (II):

or a protected derivate thereof wherein LG₁ represents a leaving groupsuch as halo, in particular bromo, with a compound of formula (III):

in the presence of a suitable catalyst and an organic base and in apolar aprotic solvent under an inert atmosphere. Suitable catalystsinclude palladium catalysts such as bis(triphenylphosphine)palladium(II) dichloride, in the presence of copper iodide. A suitable polaraprotic solvent for this transformation is DMF and a suitable inertatmosphere is nitrogen.

For synthetic processes in which the compound of formula (II) is aprotected derivative, the compound of formula (I) is revealed by anappropriate deprotection step, as is well known and practiced in theart. For example when the phenol present in the compounds of formula (I)is protected with a silyl group, for example with atert-butyldimethylsilyl group the deprotection step can be effected bytreatment with a reagent such as tetrabutylammonium fluoride in thepresence of a polar aprotic solvent such as DMF. The reaction may beperformed at a reduced temperature, such as at 0° C.

Compounds of formula (II) can be prepared by reacting a compound offormula (IV):

or a protected derivative thereof, wherein LG₁ is a leaving group, asdefined hereinabove for compounds of formula (II) and LG₂ is also aleaving group such as halo, for example a halogen atom and suitably achlorine, with a compound of formula (V):

or a protected derivative thereof, in the presence of a base and in apolar aprotic solvent.

Suitable bases for this transformation include potassium carbonate and asuitable polar aprotic solvent is DMF.

Synthetic processes include those for which is deemed advantageous toprotect the phenolic hydroxyl of the compound of formula (VII) duringthe coupling step and suitable protected derivatives include atertbutyldimethylsilyl ether and a tert-butyl ether.

Alternatively compounds of formula (II) can be prepared by reacting acompound of formula (VI):

or a protected derivative thereof, wherein LG₁, is as defined above forcompounds of formula (II) and LG₃ represents a leaving group such ashalo, in particular iodo, with a compound of formula (VII):

or a protected derivate thereof, in the presence of a suitable noblemetal catalyst, an inorganic base and in a polar protic solvent, underan inert atmosphere; followed, where appropriate, by deprotection.

A suitable catalyst is tetrakis(triphenylphosphine)palladium(0).

A suitable inorganic base is sodium carbonate and a suitable polarprotic solvent is ethanol.

The reaction may be performed at an elevated temperature, for example at85° C. for an extended period such as, for example, 3 days beforecooling to RT.

Protecting groups may be advantageous to mask chemically sensitivegroups during one or more of the reaction sequences described above, toensure that one or more of the processes are efficient. Thus if desiredor necessary, intermediate compounds may be protected by the use ofconventional protecting groups. Protecting groups and means for theirremoval are described in “Protective Groups in Organic Synthesis”, byTheodora W. Greene and Peter G. M. Wuts, published by John Wiley & SonsInc; 4^(th) Rev Ed., 2006, ISBN-10: 0471697540.

Novel intermediates are claimed as an aspect of the invention.

Advantageously, compound (I) of the present invention does not exhibitatropisomerism.

In one aspect the compound is useful in treatment, for example COPDand/or asthma.

The PI3K compounds developed to date have typically been intended fororal administration. Typically this strategy involves the optimisationof a compound's pharmacokinetic profile in order to achieve an adequateduration of action. In this way a sufficiently high drug concentrationis established and maintained between doses to provide continuousclinical benefit. An inevitable and frequently undesired consequence ofthis approach is that non-targeted body tissues, especially the liverand the gut, are likely to be exposed to pharmacologically activeconcentrations of the drug.

An alternative strategy is to design treatment regimens in which thedrug is dosed directly to the inflamed organ (for example topicaltherapy). Although this approach is not suitable for treating allchronic inflammatory conditions, it has been extensively exploited intreating lung diseases (asthma, COPD, cystic fibrosis), skin lesions(atopic dermatitis and psoriasis), nasal diseases (allergic rhinitis),eye disease (allergic conjunctivitis) and gastrointestinal disorders(ulcerative colitis).

In topical therapy, the desired efficacy can sometimes be achieved byensuring that the drug has a sustained duration of action and isretained predominantly in the target organ, thereby minimising the risksof systemic toxicity. Alternatively an appropriate formulation can beused which generates a “reservoir” of the active drug which is thenavailable to sustain the desired effects. The first approach isexemplified in the use of the anticholinergic drug tiotropium bromide(Spiriva HandiHaler®), which is administered topically to the lung as atreatment for COPD. This compound has an exceptionally high affinity forits target receptor resulting in a very slow off rate (dissociationrate) and a consequent sustained duration of action.

There is provided according to one aspect of the present disclosure useof the compound of formula (I) or a pharmaceutical formulationcontaining it, as a PI3 kinase inhibitor, for example administeredtopically to the lung.

Thus in one embodiment the compound of the present disclosure isintended for us by topical administration to the lungs in order tomaximise therapeutic benefit to patients whilst minimising the potentialfor undesirable systemic effects. It is therefore advantageous that thecompound of formula (I) is rapidly metabolised once it has reached thegeneral circulation and that the product(s) of turnover is/are lessactive than the parent molecule.

A likely principal product of first pass metabolism of the compound offormula (I) is the corresponding alcohol, compound (Ia), which wouldarise from O-demethylation, a metabolic process which is a commonfeature for structures of this chemotype. This possible metabolicproduct is significantly less active than the compound of formula (I) asa PI3K inhibitor at both the α and β subtypes (see Table 2 in theExperimental Section).

Thus, in one aspect is provided a compound of formula (Ia) or apharmaceutically acceptable salt thereof, including all stereoisomers,tautomers and isotopic derivatives thereof.

In one aspect of the disclosure the compound of the invention isparticularly suitable for topical delivery, such as topical delivery tothe lungs, in particular for the treatment of COPD.

Thus is one aspect there is provided use of a compound of the inventionfor the treatment of COPD and/or asthma, in particular COPD or severeasthma, by inhalation i.e. by topical administration to the lung.Advantageously, administration to the lung allows the beneficial effectsof the compounds to be realised whilst minimising the side-effects, forpatients.

In one embodiment the compounds are suitable for sensitizing patients totreatment with a corticosteroid.

Further, the present invention provides a pharmaceutical compositioncomprising a compound according to the disclosure optionally incombination with one or more pharmaceutically acceptable diluents orcarriers.

Diluents and carriers may include those suitable for parenteral, oral,topical, mucosal and rectal administration, and may be differentdepending on the route of administration.

In one embodiment compositions may be prepared e.g. for parenteral,subcutaneous, intramuscular, intravenous, intra-articular orperi-articular administration, particularly in the form of liquidsolutions or suspensions; for oral administration, particularly in theform of tablets or capsules; for topical e.g. pulmonary or intranasaladministration, particularly in the form of powders, nasal drops oraerosols and transdermal administration; for mucosal administration e.g.to buccal, sublingual or vaginal mucosa, and for rectal administratione.g. in the form of a suppository.

The compositions may conveniently be administered in unit dosage formand may be prepared by any of the methods well-known in thepharmaceutical art, for example as described in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,(1985).

Formulations for parenteral administration may contain as excipientssterile water or saline, alkylene glycols such as propylene glycol,polyalkylene glycols such as polyethylene glycol, oils of vegetableorigin, hydrogenated naphthalenes and the like.

Formulations for nasal administration may be solid and may containexcipients, for example, lactose or dextran, or may be aqueous or oilysolutions for use in the form of nasal drops or metered spray. Forbuccal administration typical excipients include sugars, calciumstearate, magnesium stearate, pregelatinated starch, and the like.

Compositions suitable for oral administration may comprise one or morephysiologically compatible carriers and/or excipients and may be insolid or liquid form. Tablets and capsules may be prepared with bindingagents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, orpoly-vinylpyrollidone; fillers, such as lactose, sucrose, corn starch,calcium phosphate, sorbitol, or glycine; lubricants, such as magnesiumstearate, talc, polyethylene glycol, or silica; and surfactants, such assodium lauryl sulfate. Liquid compositions may contain conventionaladditives such as suspending agents, for example sorbitol syrup, methylcellulose, sugar syrup, gelatin, carboxymethyl-cellulose, or ediblefats; emulsifying agents such as lecithin, or acacia; vegetable oilssuch as almond oil, coconut oil, cod liver oil, or peanut oil;preservatives such as butylated hydroxyanisole (BHA) and butylatedhydroxytoluene (BHT). Liquid compositions may be encapsulated in, forexample, gelatin to provide a unit dosage form.

Solid oral dosage forms include tablets, two-piece hard shell capsulesand soft elastic gelatin (SEG) capsules.

A dry shell formulation typically comprises of about 40%-60%concentration of gelatin, about a 20%-30% concentration of plasticizer(such as glycerin, sorbitol or propylene glycol) and about a 30%-40%concentration of water. Other materials such as preservatives, dyes,opacifiers and flavours also may be present. The liquid fill materialcomprises a solid drug that has been dissolved, solubilized or dispersed(with suspending agents such as beeswax, hydrogenated castor oil orpolyethylene glycol 4000) or a liquid drug in vehicles or combinationsof vehicles such as mineral oil, vegetable oils, triglycerides, glycols,polyols and surface-active agents.

Suitably the compound of formula (I) is administered topically to thelung. Hence in one embodiment there is provided a pharmaceuticalcomposition comprising a compound of the disclosure optionally incombination with one or more topically acceptable diluents or carriers.Topical administration to the lung may be achieved by use of an aerosolformulation. Aerosol formulations typically comprise the activeingredient suspended or dissolved in a suitable aerosol propellant, suchas a chlorofluorocarbon (CFC) or a hydrofluorocarbon (HFC). Suitable CFCpropellants include trichloromonofluoromethane (propellant 11),dichlorotetrafluoromethane (propellant 114), and dichlorodifluoromethane(propellant 12). Suitable HFC propellants include tetrafluoroethane(HFC-134a) and heptafluoropropane (HFC-227). The propellant typicallycomprises 40%-99.5% e.g. 40%-90% by weight of the total inhalationcomposition. The formulation may comprise excipients includingco-solvents (e.g. ethanol) and surfactants (e.g. lecithin, sorbitantrioleate and the like). Aerosol formulations are packaged in canistersand a suitable dose is delivered by means of a metering valve (e.g. assupplied by Bespak, Valois or 3M).

Topical administration to the lung may also be achieved by use of anon-pressurised formulation such as an aqueous solution or suspension.This may be administered by means of a nebuliser. Topical administrationto the lung may also be achieved by use of a dry-powder formulation. Adry powder formulation will contain the compound of the disclosure infinely divided form, typically with a mass mean diameter (MMAD) of 1-10μm. The formulation will typically contain a topically acceptablediluent such as lactose, usually of large particle size e.g. a mass meandiameter (MMAD) of 100 μm or more. Example dry powder delivery systemsinclude SPINHALER®, DISKHALER®, TURBOHALER®, DISKUS®, SKYEHALER®,ACCUHALER® and CLICKHALER®.

In one embodiment a compound of the present invention is provided as amicronized dry powder formulation, for example comprising lactose of asuitable grade, filled into a device such as DISKUS.

The compounds according to the disclosure are intended to havetherapeutic activity. In a further aspect, the present inventionprovides a compound of the disclosure for use as a medicament.

The compounds according to the disclosure may also be useful in thetreatment of respiratory disorders including COPD (including chronicbronchitis and emphysema), asthma, paediatric asthma, cystic fibrosis,sarcoidosis, idiopathic pulmonary fibrosis, allergic rhinitis, rhinitis,sinusitis and virally induced exacerbations of any one of the same,repiratory virus infection, especially asthma, chronic bronchitis andCOPD.

Respiratory viruses include influenza, respiratory syncytical virus,human parainfluenza virus, SARS coronavirus and adenoviruses.

The compound of the disclosure may also re-sensitise the patient'scondition to treatment with a corticosteroid, when previously thepatient's condition had become refractory to the same.

In one embodiment of the invention a dose of the present compound isemployed that is equal to that suitable for use as a monotherapy butadministered in combination with a corticosteriod.

In one embodiment a dose of the compound of formula (I) that would besub-therapeutic as a single agent is employed, in combination with acorticosteriod, thereby restoring patient responsiveness to the latter,in instances where the patient had previously become refractory to thesame.

Additionally, the compound of the disclosure may exhibit anti-viralactivity and, for example, prove useful in the treatment of viralexacerbations of inflammatory conditions such as asthma and/or COPD.

The compound of the present disclosure may also be useful in theprophylaxis, treatment or amelioration of disease or the complicationsof disease associated with influenza virus, rhinovirus and/orrespiratory syncytial virus.

In one embodiment there is provided a compound of formula (I) for use inthe treatment or prevention of viral infection or inflammatorycomplications induced by viral infection.

The compound of formula (I), according to the disclosure is alsoexpected to be useful in the treatment of certain conditions which maybe treated by topical or local therapy including allergicconjunctivitis, conjunctivitis, allergic dermatitis, contact dermatitis,psoriasis, ulcerative colitis, inflamed joints secondary to rheumatoidarthritis or osteoarthritis.

In one embodiment the compound of formula (I) is considered useful inthe treatment of Hepatitis C and/or HIV, when administered by anappropriate route. Appropriate routes of administration may includeoral, intravenous injection or infusion.

In one embodiment the compound of formula (I) for the treatment ofHepatitis C is delivered to the blood pre-entry to the liver.

The compound of the disclosure is also expected to be useful in thetreatment of certain other conditions including rheumatoid arthritis,pancreatitis, cachexia, inhibition of the growth and metastasis oftumours including non-small cell lung carcinoma, breast carcinoma,gastric carcinoma, colorectal carcinomas and malignant melanoma.

In one embodiment the presently disclosed compound and pharmaceuticalformulations comprising the same are useful in the treatment orprevention of cancer, in particular lung cancer, especially by topicaladministration to the lung.

Thus, in a further aspect, the present invention provides the compoundas described herein for use in the treatment of one or more of the abovementioned conditions.

In a further aspect, the present invention provides use of the compoundas described herein for the manufacture of a medicament for thetreatment of one or more of the above mentioned conditions.

In a further aspect, the present invention provides a method oftreatment of the above mentioned conditions which comprisesadministering to a subject an effective amount of the compound of thedisclosure or a pharmaceutical composition thereof.

The compound described herein may also be used in the manufacture of amedicament for the treatment of one or more of the above-identifieddiseases.

The word “treatment” is intended to embrace prophylaxis as well astherapeutic treatment.

The compound of the disclosure may also be administered in combinationwith one or more other active ingredients e.g. active ingredientssuitable for treating the above mentioned conditions. For examplepossible combinations for treatment of respiratory disorders includecombinations with corticosteroids (e.g. budesonide, beclomethasonedipropionate, fluticasone propionate, mometasone furoate, fluticasonefuroate), beta agonists (e.g. terbutaline, salbutamol, salmeterol,formoterol, indacaterol) and/or xanthines (e.g. theophylline),musacarinic antagonists, (e.g. ipratropium) and/or a p38 MAP kinaseinhibitor.

In one embodiment the compound of the disclosure is administered incombination with an antiviral agent, for example acyclovir, oseltamivir,relenza or interferon.

In one embodiment the combination of active ingredients areco-formulated.

In one embodiment the compound of the present disclosure isco-formulated with a corticosteriod as a formulation for inhalation, forexample for use in maintenance therapy of COPD or lung cancer includingprevention of the latter.

In one embodiment the combination of active ingredients is simplyco-administered.

In one embodiment there is provided a combination product comprising:

-   (A) a compound of the present disclosure; and-   (B) a further active ingredient e.g. selected from corticosteroids,    beta agonists, xanthenes, musacarinic antagonists and p38 MAP kinase    inhibitors.    wherein each of components (A) and (B) is formulated in admixture    with a pharmaceutically-acceptable diluent or carrier.

In one embodiment there is provided a compound of formula (I) accordingto claim 1 for use as a medicament to be administered in combinationwith one or more further active ingredients e.g. selected fromcorticosteroids, beta agonists, xanthenes, musacarinic antagonists andp38 MAP kinase inhibitors.

In one embodiment the compound of the disclosure is administered byinhalation and a corticosteroid is administered orally or by inhalationeither in combination or separately.

EXPERIMENTAL SECTION

Abbreviations used herein are defined below (Table 1). Any abbreviationsnot defined are intended to convey their generally accepted meaning.

TABLE 1 Abbreviations aq aqueous Ac acetyl ACD acid citrate dextrose ATPadenosine-5′-triphosphate BALF bronchoalveolae lavage fluid br broad BSAbovine serum albumin COPD chronic obstructive pulmonary disease CXCL1CXC motif chemokine ligand 1 CXCL8 CXC motif chemokine ligand 8 ddoublet DCM dichloromethane DMAP 4-dimethylaminopyridine DMSO dimethylsulfoxide EDC•HCl 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride ELISA enzyme linked immunosorbent assay (ES⁺) electrosprayionization, positive mode Et ethyl EtOAc ethyl acetate EtOH ethanol FACSfluorescence-activated cell sorting FCS foetal calf serum FITCfluorescein isothiocyanate FP fluticasone propionate HPLC-MS highperformance liquid chromatography mass spectrometry hr hour(s) HRPhorseradish peroxidase IFNγ Interferon gamma i-n intra-nasal i-tintra-tracheal IL-4 interleukin 4 IL-5 interleukin 5 IL-13 interleukin13 IL-17 Interleukin 17 LPS lipopolysaccharide IPA isopropanol[propan-2-ol] M molar (M + H)⁺ protonated molecular ion MCP-1 monocytechemoattractant protein MDA malondialdehyde Me methyl MeOH methanol MHzmegahertz min minute(s) MOMA-2 anti-monocyte and macrophage antibody MTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide m/zmass-to-charge ratio NH₄OAc ammonium acetate nm nanometre NMR nuclearmagnetic resonance (spectroscopy) OVA ovalbumin PBMC peripheral bloodmononuclear cell(s) PBS phosphate buffered saline Pd₂(dba)₃tris(dibenzylideneacetone)dipalladium(0) PdCl₂(PPh₃)₂bis(triphenylphosphine)palladium(II) dichloride Ph phenyl PIP2phosphatidylinositol 4,5-biphosphate PIP3 phosphatidylinositol3,4,5-triphosphate PMA phorbol myristate acetate po by oraladministration PPh₃ triphenylphosphine q quartet quin quintet R^(t)retention time RT room temperature RP HPLC reverse phase highperformance liquid chromatography s singlet SDS sodium dodecyl sulfateSEM standard error of the mean t triplet TMB3,3′,5,5′-tetramethylbenzidine TNFα tumour necrosis factor alpha TR-FRETtime-resolved fluorescence resonance energy transfer

General Procedures

All starting materials and solvents were obtained either from commercialsources or prepared according to the literature citation. Unlessotherwise stated all reactions were stirred. Organic solutions wereroutinely dried over anhydrous magnesium sulfate.

Column chromatography was performed on pre-packed silica (230-400 mesh,40-63 μm) cartridges using the amount indicated.

Analytical Methods Analytical LCMS

Analytical LCMS was carried out using a Waters Xselect CSH C₁₈ 3.5 μmcolumn (4.6×50 mm) with a flow rate of 2.5-4.5 mL min⁻¹ eluting with aH₂O-MeCN gradient containing 0.1% v/v formic acid over 4 min. Gradientinformation: 0-3.00 min, ramped from 95% H₂O-5% MeCN to 5% H₂O-95% MeCN;3.00-3.01 min, held at 5% H₂O-95% MeCN, flow rate increased to 4.5 mLmin⁻¹; 3.01 3.50 min, held at 5% H₂O-95% MeCN; 3.50-3.60 min, returnedto 95% H₂O-5% MeCN, flow rate reduced to 3.50 mL min⁻¹; 3.60-3.90 min,held at 95% H₂O-5% MeCN; 3.90-4.00 min, held at 95% H₂O-5% MeCN, flowrate reduced to 2.5 mL min⁻¹. Sample containing fractions were detectedby their UV absorbance at 254 nm. Mass spectra of the eluted peaks weremeasured using an Agilent 6120 quadrupole mass spectrometer operating inmixed positive and negative ion electrospray modes.

¹H NMR Spectroscopy

¹H NMR spectra were acquired on a Bruker Avance III spectrometer at 400MHz using residual undeuterated solvent as reference and unlessspecified otherwise were run in DMSO-d₆.

Intermediate A:2-((4-Amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-bromo-3-(2-(trifluoromethyl)benzyl)quinazolin-4(3H)-one

To a stirred mixture of 2-bromo-6-(2-chloroacetamido)benzoic acid[King-Underwood et al., WO2011/048111], (50.0 g, 171 mmol),(2-(trifluoromethyl)phenyl)methanamine (29.4 mL, 205 mmol) andtriethylamine (34 mL, 430 mmol) in toluene (1.2 L) at −1° C. was added asolution of phosphorus trichloride (37 mL, 430 mmol) in toluene (100 mL)dropwise over 1 hr, during which time the internal temperature wasmaintained below 5° C. The reaction mixture was heated to reflux for 2.5hr and the resulting suspension was then filtered whilst still hot. Thefiltrate was retained and the collected solid was suspended in freshtoluene (100 mL), and was heated to 90° C. with vigorous stirring. Thesolid was removed by filtration and the organic extracts containing thecrude product were combined.

A second batch of this material was prepared by repeating the reactionunder identical conditions on the same scale. The combined filtratesfrom both reactions were evaporated in vacuo and the residue wastriturated with IPA (2×400 mL). The crude material so obtained was driedin vacuo to afford5-bromo-2-(chloromethyl)-3-(2-(trifluoromethyl)benzyl)quinazolin-4(3H)-oneas an off-white solid (100 g, 90% purity by HPLC, 61%); R^(t) 2.70 min;m/z 431/433 (M+H)⁺ (ES⁺).

To a solution of the quinazolinone, obtained as described above, (100 g,90% pure, 210 mmol) and 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine (50.4g, 193 mmol) in DMF (600 mL) at RT was added potassium carbonate (80.0g, 580 mmol) and after 18 hr the reaction mixture was poured into water(1.2 L). The resulting precipitate was collected by filtration, and waswashed sequentially with water (500 mL), with EtOAc (600 mL) and finallywith Et₂O (400 mL). The resulting cake was dried in vacuo to afford thetitle compound, Intermediate A, as an off white solid (115 g, 89%);R^(t) 2.28 min, m/z 656/658 (M+H)⁺ (ES⁺).

Intermediate B:2-((4-Amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-bromo-3-(2-(trifluoromethyl)benzyl)quinazolin-4(3H)-one

A solution of Intermediate A (54.4 g, 83.0 mmol),(3-fluoro-5-hydroxyphenyl)boronic acid (15.5 g, 99.0 mmol) and K₃PO₄(19.1 g, 83.0 mmol) in 1-butanol (900 mL) was sparged with nitrogen atRT for 20 min. The mixture was treated with PPh₃ (3.26 g, 12.4 mmol) andwith Pd₂(dba)₃ (1.90 g, 2.07 mmol) and was sparged with nitrogen for anadditional 10 min and was then heated to 90° C. under a flow ofnitrogen. After 40 hr the mixture was cooled to 70° C. and water (250mL) was added dropwise. The mixture was cooled to 50° C. for 3 hr andthen to RT for 3 days during which time a beige precipitate formed. Thesolid was collected by filtration, washed with 1-butanol (2×100 mL) andwith water (2×100 mL) and was then dried in vacuo at 40° C. to affordthe title compound, Intermediate B, as an off-white solid (35.2 g, 65%);R^(t) 2.25 min, m/z 640/642 (M+H)⁺ (ES⁺).

Compound (I):2-((4-Amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-(3-(2-(2-methoxyethoxy)ethoxy)prop-1-yn-1-yl)-3-(2-(trifluoromethyl)benzyl)quinazolin-4(3H)-one

A suspension of Intermediate B (35.2 g, 55.0 mmol),3-(2-(2-methoxyethoxy)ethoxy)prop-1-yne [King-Underwood et al.,WO2011/048111], (17.4 g, 110 mmol), PdCl₂(PPh₃)₂ (3.86 g, 5.50 mmol) andcopper(I) iodide (1.05 g, 5.50 mmol) in a mixture of Et₂NH and DMF (4:1v/v, 820 mL) was sparged with nitrogen at RT for 10 min. The mixture washeated to 65° C. for 1.5 hr and was then cooled to RT. The volatileswere evaporated in vacuo and the residue was partitioned between EtOAc(600 mL) and sat. aq. NH₄OAc (650 mL). The aq layer was separated andwas extracted with EtOAc (300 mL) and the combined organic layers wereevaporated in vacuo to afford a dark brown viscous oil. Methanol (200mL) was added and the mixture was stirred at RT for 16 hr. A yellowprecipitate was formed which was collected by filtration and washed withMeOH (100 mL). The resulting solid was purified by flash columnchromatography in two separate batches (SiO₂, 330 g, MeOH in DCM, 0-6%,gradient elution). The purified materials were taken up together, in amixture of DCM/MeOH (10:1 v/v) to give a homogeneous solution which wasthen evaporated and dried in vacuo to afford the title compound,Compound (I), as an off-white solid (20.1 g, 51%); Rt 2.15 min, m/z 718(M+H)⁺ (ES⁺); ¹H NMR δ: 3.19 (3H, s), 3.35-3.38 (2H, overlapping m),3.44-3.49 (4H, overlapping m), 3.60-3.63 (2H, overlapping m), 4.37 (2H,s), 5.49 (2H, s), 5.76 (2H, s), 6.42 (1H, d), 6.65 (1H, m), 6.73 (1H,m), 6.79 (1H, m), 7.15 (1H, t), 7.28 (1H, t), 7.52 (1H, d), 7.65-7.69(2H, overlapping m), 7.82 (1H, m), 8.12 (1H, s), 10.15 (1H, s)

Compound (Ia):2-((4-Amino-3-(3-fluoro-5-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-(3-(2-(2-hydroxyethoxy)ethoxy)prop-1-yn-1-yl)-3-(2-(trifluoromethyl)benzyl)quinazolin-4(3H)-one

A suspension of Intermediate B (190 mg, 0.297 mmol),2-(2-(prop-2-yn-1-yloxy)ethoxy) ethanol [King-Underwood et al.,WO2011/048111] (257 mg, 0.890 mmol), PdCl₂(PPh₃)₂ (208 mg, 0.297 mmol)and copper(I) iodide (57 mg, 0.30 mmol) in a mixture of Et₂NH and DMF(4:1 v/v, 7.5 mL) was degassed with N₂ and was then heated to 60° C. for16 hr. The reaction mixture was cooled to RT and was evaporated in vacuoonto silica gel and purified by flash column chromatography (SiO₂, 12 g,MeOH in DCM, 0-5%, gradient elution) to afford the title compound,Compound (Ia), as a pale tan solid (30 mg, 14%); Rt 1.88 min, m/z 704(M+H)⁺ (ES⁺); ¹H NMR δ: 3.36-3.50 (6H, overlapping m), 3.61-3.63 (2H,overlapping m), 4.37 (2H, s), 4.58 (1H, m), 5.48 (2H, s), 5.76 (2H, s),6.41 (1H, d), 6.64 (1H, m), 6.72 (1H, d), 6.78 (1H, s), 7.14 (1H, t),7.27 (1H, t), 7.52 (1H, d), 7.65-7.71 (2H, overlapping m), 7.82 (1H, t),8.13 (1H, s), 10.19 (1H, br s).

The additional complexity and consequences for drug developmentresulting from atropisomerism are analogous to those arising from othersources of molecular isomerism such as the presence of a stereogeniccentre. This property renders such molecules both chiral, and unlessresolved, a racemic mixture; the components of which could possessdifferent pharmacological and toxicological profiles. This feature islikely to significantly increase downstream development costs for suchmolecules, and the absence of atropisomerism in compound (I) istherefore a highly desirable and advantageous property.

Biological Testing: Experimental Methods Enzyme Inhibition Assay

The PI3K enzymes catalyse the phosphorylation of phosphatidylinositol4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3)in the presence of ATP and Mg²⁺ ions. The PIP3 product can be detectedby displacement of biotin-PIP3 from energy transfer complexes consistingof europium labelled anti-GST monoclonal antibody, a GST-taggedPleckstrin homology (PH) domain, biotinylated PIP3 andstreptavidin-allophycocyanin (APC) by the time-resolved fluorescenceresonance energy transfer (TR-FRET) (HTRF®PI3K enzyme assay, Millipore).Excitation, at 330 nm, of europium in the complex results in an energytransfer to the APC and a fluorescent emission at 665 nm althougheuropium itself emits at its characteristic wavelength of 620 nm. ThePIP3 product formed by PI3K activity displaces biotin-PIP3 from thecomplex and results in a loss of energy transfer (decreasing signal).

The compound to be tested was added, at the desired finalconcentrations, to a mixture of PIP2 substrate and a recombinant PI3Kenzyme (either α, β or δ isoforms, ex Millipore, or γ isoform[p110γ+p101 construct], ex United States Biological, Swampscott, Mass.)and the mixture incubated for 2 hr at RT. Following this incubationperiod, ATP (10 μM) was added to the enzyme/compound/PIP2 substratemixture and the resulting mixture was incubated for a further 30 min atRT. A stopping solution containing biotinylated PIP3 and the detectionmix containing the GST tagged GRP1 pleckstrin homology (PH) domain andfluorophores were then added and the mixture was incubated at RT for15-18 hr, prior to detection in a fluorescence microplate reader(Synergy 4, BioTek UK, Bedfordshire, UK).

The results were calculated according to the formula: APC signal(emission at 665 nm)/europium signal (emission at 620 nm)×10⁴. Thepercentage inhibition of each reaction was calculated relative to DMSOtreated control, and the 50% inhibitory concentration (IC₅₀ value) thencalculated from the concentration-response curve.

PI3K δ Cell Based Assay

As a means of assessing PI3K δ activation in response to stimuli, thephosphorylation status of the protein, Akt, a downstream product ofPI3Kδ, signaling was determined.

U937 cells, obtained from a human, leukemic, monocyte lymphoma cellline, were differentiated to macrophage-type cells by incubation withPMA (100 ng/mL) for 48 to 72 hr. Cells were then pre-incubated witheither the test compound or vehicle for 2 hr and were then stimulatedbriefly by exposure to H₂O₂ (10 mM, 5-7 min) and the reaction stopped byreplacing the media with 4% formaldehyde solution. Endogenous peroxideactivity and formaldehyde were inactivated by incubating with quenchingbuffer (0.1% sodium azide, 1% H₂O₂ in PBS with 0.1% Triton X-100) for 20min. The cells were washed with buffer (PBS containing 0.1% TritonX-100) and were incubated with blocking solution (1% BSA in PBS) for 1hr and were then re-washed with buffer and incubated overnight witheither anti-pAkt antibody or anti-pan-Akt antibody (both from CellSignaling Technology). After washing with buffer (PBS containing 0.1%Triton X-100), cells were incubated with an HRP-conjugated secondaryantibody (Dako) and the resultant signal was determined colorimetrically(OD: 450 nm with a reference wavelength of 655 nm) using TMB substrate(substrate reagent pack supplied by R&D Systems, Inc.).

This reaction was stopped by the addition of H₂SO₄ solution (100 μL).Cells were then washed with buffer (PBS containing 0.1% Triton X-100)and 5% crystal violet solution (100 μL) was applied for 30 min. Afterwashing with buffer (PBS containing 0.1% Triton X-100) 1% SDS (100 μL)was added to each well and the plates were shaken lightly for 1 hr priorto measuring the absorbance at 595 nm (Varioskan® Flash, Thermo-FisherScientific). The measured OD₄₅₀₋₆₅₅ readings were corrected for cellnumber by dividing the OD₄₅₀₋₆₅₅ by the OD₅₉₅ readings. The ratio ofpAkt signal to total Akt signal was used to quantitate the extent ofPI3K δ activation. The percentage inhibition for each well wascalculated relative to a 10 μg/mL standard control (LY294002) set to100% inhibition versus H₂O₂ only controls as 0% inhibition. The IC₅₀values were calculated from the concentration-response curves generatedby the serial dilutions of the test compounds.

PI3K γ Cell Based Assay

As a means of assessing the activation of PI3K γ in response to stimuli,the phosphorylation status of the protein, Akt, a downstream product ofPI3K γ signalling, was determined following stimulation with MCP-1.

U937 cells were differentiated to macrophage-type cells by incubationwith PMA (100 ng/mL) for 48 to 72 hr. Cells were then pre-incubated witheither the test compound or vehicle for 2 hr and were then stimulatedbriefly with MCP-1 (10 nM, 1 min) and the reaction stopped by replacingthe media with 4% formaldehyde solution. Endogenous peroxide activityand formaldehyde were inactivated by incubating with quenching buffer(0.1% sodium azide, 1% H₂O₂ in PBS with 0.1% Triton X-100) for 20 min.The cells were washed with buffer (PBS containing 0.1% Triton X-100) andwere incubated with blocking solution (1% BSA in PBS) for 1 hr and werethen re-washed with buffer and incubated overnight with either anti-pAktantibody or anti-pan-Akt antibody (both from Cell Signaling Technology).After washing with buffer (PBS containing 0.1% Triton X-100), cells wereincubated with an HRP-conjugated secondary antibody (Dako) and theresultant signal was determined colorimetrically (OD: 450 nm with areference wavelength of 655 nm) using TMB substrate (substrate reagentpack supplied by R&D Systems, Inc.).

This reaction was stopped by addition of 1N H₂SO₄ solution (100 μL).Cells were then washed with buffer (PBS containing 0.1% Triton X-100)and 5% crystal violet solution (100 μL) was applied for 30 min. Afterwashing with buffer (PBS containing 0.1% Triton X-100) 1% SDS (100 μL)was added to each well and the plates were shaken lightly for 1 hr priorto measuring the absorbance at 595 nm (Varioskan® Flash, Thermo-FisherScientific). The measured OD₄₅₀₋₆₅₅ readings were corrected for cellnumber by dividing the OD₄₅₀₋₆₅₅ by the OD₅₉₅ readings. The ratio ofpAkt signal to total Akt signal was used to quantitate the extent ofPI3K γ activation. The percentage inhibition for each well wascalculated relative to a 10 μg/mL standard control (LY294002) set to100% inhibition versus MCP-1 only controls as 0% inhibition. The IC₅₀values were calculated from the concentration-response curves generatedby the serial dilutions of the test compounds using XL-Fit (idbs,Guildford, UK).

Superoxide Anion Production Assay

As a means of assessing PI3Kδ dependent cell function, superoxide anionproduction in IFNγ-primed U937 cells was evaluated by achemiluminescence assay. The U937 cells (purchased from ATCC, Manassas,Va.) were maintained in RPMI 1640 (Invitrogen Ltd., Paisley, UK) with10% FCS at 37° C. The cells were suspended at a density of 10⁷ cells/mLin 40 mL 10% FCS RPMI 1640, and treated with 20 μL of 100 μg/mL IFNγsolution (final concentration: 50 ng/mL), and incubated at 37° C., 5%CO₂ for 4 days.

IFNγ-primed U937 cells were seeded at 0.2×10⁶ cells/well in a 96-wellplate, and pre-incubated with test compounds for 2 hr in starvationmedia (0.5% FCS RPMI1640-phenol red free). Zymosan A (10 mg) fromSaccharomyses cerevisiae (ex Sigma-Aldlich) was resuspended in 1 mL of150 mM NaCl and boiled at 100° C. for 15 min. After boiling, Zymosanparticles were wash with 1 mL of PBS twice, and incubated with 0.5 mL ofBioParticles™ opsonized reagents (Life technologies) for 60 min at 37°C. Cells were treated with a mixture of Zymosan particle solution (10μL), assay buffer (85 μL), luminol (2.5 μL) and enhancer solution (2.5μL), which are all provided (apart from Zymosan) in the Superoxide AnionAssay Kit (# CS1000, Sigma-Aldrich Ltd, Poole, UK). Chemiluminescenceindicating released superoxide anion was measured every 15 min up to 60min by luminometric measurement (Varioskan® Flash, Thermo-FisherScientific).

The data at 60 min after incubation was used for analysis. Thepercentage inhibition for each well was calculated relative to a 10μg/mL IC87114, a standard PI3Kδ inhibitor, set to 100% inhibition versuscontrol as 0% inhibition. The relative EC₅₀ values were calculated fromthe concentration-response curves generated by the serial dilutions ofthe test compounds using XL-Fit (idbs, Guildford, UK).

Cytostim-Induced Cytokine Production in PBMCs

As a means of assessing PI3Kδ-dependent cell function, cytostim-inducedIL-4, IL-5, IL-13 and IFNγ production in PBMCs were evaluated by Luminexmultiplex assay. All healthy volunteers were recruited by QuintilesLimited (London, UK), and blood samples were delivered to Respivert Ltd.This study was approved by the local Ethics Committee, and all subjectsgave written informed consent.

PBMC suspensions (200 μL; 2×10⁶ cells/mL) were added to a 96-well plate.Cells were treated with either test compounds in neat DMSO or DMSO asvehicle (2 μL) and incubated at RT for 1 hr. Cytostim (Miltentyi Biotec,Surrey, UK) was introduced at a ratio of 1:50 and the cells wereincubated for 20 hr (37° C.; 5% CO₂). Plates were spun at 500×g for 5min and the supernatant collected. A high sensitivity cytokine magneticbead kit (#HSCYTMAG-605K, Millipore, Watford, UK) was used to measurethe four analytes (IL-4, IL-5, IL-13 and IFNγ) by Luminex as follows:The magnetic antibody beads were multiplexed and incubated in a 96-wellplate with standard, media only or sample (50 μL) overnight with shakingat 4° C. After washing twice with Millipore wash buffer provided in thekit using a magnetic plate washer, the beads were incubated for 1 hrwith detection antibody (50 μL) with shaking at RT. Astreptavidin-phycoerythrin solution provided in the kit was added for 30min with shaking at RT. After washing, the beads were re-suspended insheath fluid (150 μL) and analysed immediately. The Luminex system wasset up to count 50 beads and the amount of each analyte in thesupernatant was calculated against a standard curve. The IC₅₀ valueswere determined from concentration-inhibition curves using XL-Fit (IDBS,Guildford, UK)

Chemotaxis to MCP1

As a means of assessing PI3K γ dependent cell function, THP1 cellchemotaxis to MCP-1 was evaluated using a 48 well-chemotaxis chamber.THP1 cells from a human leukemic monocyte lymphoma cell line, (purchasedfrom ATCC Manassas, Va.) were maintained in RPMI 1640 (Invitrogen Ltd.,Paisley, UK) with 10% FCS at 37° C. Cells were re-suspended in 0.5%BSA/RPMI1640 (2×10⁶ cells/mL) and incubated for 10 min at 37° C., 5%CO₂. Cell suspension aliquots (500 μL) were then treated either withcompounds in neat DMSO or with DMSO as vehicle (2.5 μL) for 1 hr (37°C., 5% CO₂).

MCP1 (50 nM) was prepared in 0.5% BSA/RPMI1640. MCP1 solution (50 μL)was added to each well in the bottom plate of a 48-well chemotaxischamber (AP48, NeuroProbe Inc., Gaithersburg, Md.). A polycarbonatemembrane (8 μm) was mounted on the bottom chamber, and then the topplate was mounted on the bottom chamber and filter membrane. Cellsuspension aliquots (50 μL) were treated with compounds or vehiclebefore being added into the top chamber carefully, and 0.5% BSA RPMI1640(50 μL) was applied on top. The chamber was then left for 2 hr (37° C.,5% CO₂). The membrane was then carefully removed and sample (25 μL) fromthe bottom chamber was transferred to a new 96 well plate.

A solution of MTT (50 μL) in 10% FCS phenol red-free RPMI1640 was addedto each well, and the plate was incubated for 2 hr (37° C., 5% CO₂).Neat DMSO (100 μL) was added to each well to extract formazan formedfrom MTT and the plates were shaken lightly for 1 hr prior to measuringthe absorbance at 595 nm (Varioskan® Flash, Thermo-Fisher Scientific).Values were compared to inhibition by AS604850 (10 μg/mL), a selectivePI3Kγ inhibitor, to calculate the relative percent inhibitions. RelativeEC₅₀ values were determined from concentration-inhibition curves usingXL-Fit (idbs, Guildford, UK).

CXCL8 Release from Neutrophils Obtained from COPD Patients

The effects of treatment on CXCL8 release from neutrophils obtained fromCOPD patients were evaluated by an ELISA assay. All patients wererecruited by Quintiles Limited (London, UK), and blood samples weredelivered to Respivert Ltd. This study was approved by the local EthicsCommittee, and all subjects gave written informed consent. Whole blood(30 mL) was mixed gently with ACD (5 mL; comprising: 7.36 g citric acid,14.71 g sodium citrate, 9.91 g dextrose in 250 ml sterile, doubledistilled water) and 6% dextran (15 mL; diluted in 0.9% NaCl) to removethe red blood cells. The tubes were incubated at RT for 45 min, and thesupernatant (white cell rich fraction) was then collected, leaving thered blood cells.

This fraction was centrifuged (10 min at 1200 rpm, 4° C.) with lowbraking. The supernatant was aspirated and the pellet was re-suspendedin ice-cold, sterile double distilled H₂O (10 mL), and 0.6 M KCl (4 mL)was added 30 seconds later. The cell suspension was diluted with sterilePBS (to a final volume of 50 mL) and then centrifuged at 1500 rpm for 5min. The supernatant was aspirated and the pellet re-suspended in PBS(2.5 mL), and two tubes from the same donor were pooled into one.

This cell suspension was carefully layered on top of 5 mL ofFicoll-Paque™ premium (GE Healthcare Bio Science AB, Uppsala, Sweden)using a Pasteur pipette, and then centrifuged (30 min at 1500 rpm withlow braking). The isolated neutrophils at the bottom of the tubes werere-suspended in RPMI-1640 medium (Gibco, Paisley, UK) containing 5% FCSand seeded at a density of 4×10⁵ cells/well in a 96 well plate. Thecells were incubated for 30 min (37° C.; 5% CO₂) before treatment wasbegun.

Neutrophils were pre-incubated with test compounds or with DMSO vehiclefor 1 hr and then stimulated with TNFα (10 ng/mL). Cell free supernatantwas collected at 3 hr after TNFα stimulation, and CXCL8 was measured byELISA using DuoSet ELISA development kit (R&D systems, Abingdon, UK).The IC₅₀ values reported were determined from concentration-inhibitioncurves using XL-Fit (IDBS, Guildford, UK).

MTT Assay

PMA-differentiated U937 cells were pre-incubated with test compound (10μg/mL) or vehicle for 4 hr in 5% FCS or 10% FCS for 24 hr. Thesupernatant was replaced with new media (200 μL) and MTT stock solution(10 μL, 5 mg/mL) was added to each well. After 1 hr incubation, themedia were removed, 200 μL of DMSO was added to each well and the plateswere shaken lightly for 1 hr prior to reading the absorbance at 550 nm.The percentage loss of cell viability was calculated for each wellrelative to vehicle (0.5% DMSO)-treatment.

In Vivo Screening: Pharmacodynamics and Anti-Inflammatory ActivityLPS-Induced Airway Neutrophil Accumulation in Mice

Non-fasted BALB/c mice (6-8 weeks old) were dosed with vehicle or withtest compound by intra-tracheal administration (dose volume 20 μL) attime points T=−2 hr, −8 hr, or −12 hr with respect to the start of LPStreatment. The LPS was prepared in a solution of 0.5 mg/mL andaerosolised using a De Vibliss ultrasonic nebuliser 2000 (7 mL during 30min exposure). At eight hr after LPS challenge, the trachea wascannulated and bronchoalveolar lavage fluid (BALF) extracted by infusingand then withdrawing PBS (1 mL) into the lungs via the trachealcatheter. This procedure was repeated to give a yield of approximately 2mL lavage fluid. Total cell numbers in the BALF samples were measuredusing a haemocytometer. Cytospin smears of the BALF samples wereprepared by centrifugation at 1200 rpm for 2 min at RT and stained usinga DiffQuik stain system (Dade Behring) for differential cell counts.Cells were counted using oil immersion microscopy. Data are expressed asneutrophil number of cells per mL of BALF (mean±S.E.M).

LPS-Induced Airway Neutrophil Accumulation in Rats

Non-fasted rats were dosed with either vehicle or the test compound byintra-tracheal administration (dose volume 100 μL) at time points T=−2h, −8 h, or −12 h with respect to the start of LPS treatment. The LPSsolution (0.3 mg/mL) was aerosolised using a De Vibliss ultrasonicnebuliser 2000 (7 mL during 30 min). At eight hr after LPS challenge,the trachea was cannulated and bronchoalveolar lavage fluid (BALF)extracted by infusing and then withdrawing PBS (1 mL) into the lungs viathe tracheal catheter. This procedure was repeated to provideapproximately 2 mL of lavage fluid.

Total cell numbers in the BALF samples were measured using a Countessautomated cell counter (Invitrogen). Cytospin smears of the BALF sampleswere prepared by centrifugation at 1200 rpm for 2 min at RT and stainedusing a DiffQuik stain system (Dade Behring) for differential cellcounts. Cells were counted using oil immersion microscopy. Data areexpressed as neutrophil number of cells per mL of BALF (mean±S.E.M).

Ovalbumin-Induced Airway Eosinophil and Neutrophil Accumulation in Mice

BALB/c mice (6-8 weeks old) were immunized with OVA (10 μg/mouse i.p.)on days 0 and 7. In order to elicit a local inflammatory response in thelung, mice were repeatedly challenged between days 13-15 with anebulised solution of ovalbumin (10 mg/mL, 30 min exposure, De VilibissUltraneb 2000). On day 17 each animal received via intra-trachealadministration of either vehicle or test compound 2 hr prior to thefinal OVA challenge. Animals were anaesthetized 8 hr later beforeundergoing a tracheotomy. BAL was obtained by instilling PBS (1 mL) intothe lungs, which was then withdrawn. This procedure was repeated toprovide approximately 2 mL of lavage fluid. Total cell numbers in theBALF samples were measured using a haemocytometer. Cytospin smears ofthe BALF samples were prepared by centrifugation at 200 rpm for 5 min atRT and stained using a DiffQuik stain system (Dade Behring) fordifferential cell counts. Cells were counted using oil immersionmicroscopy. Data is expressed as differential number of cells per mL ofnasal lavage fluid (mean±S.E.M).

Poly-I:C-Induced Cell Accumulation in Mice

Specific pathogen-free A/J mice (males, 5 weeks old) were dosed withpoly (I:C)-LMW (1 mg/mL, 40 μL) (InvivoGen, San Diego, Calif., USA)intranasally twice daily for 3 days under anaesthasia (3% isoflurane).Test substances were dosed intra-nasally (50 μL in 10% DMSO/isotonicsaline vehicle) 2 hr before each poly-I:C treatment. At 24 hr after thelast poly-I:C challenge, animals were anesthetized, the trachea wascannulated and bronchial alveolar lavage (BAL) was obtained byinstilling into and then withdrawing from the lungs, isotonic saline(100 mL/kg). Total cell numbers in the BALF samples were measured usinga haemocytometer under a phase-contrast microscope.

The proportions of alveolar macrophages and neutrophils were determinedby FACS analysis using anti-mouse MOMA2-FITC (macrophage) or anti-mouse7/4-FITC (neutrophil). Cells were suspended in PBS and incubated withanti-MOMA2-FITC (2 μg/mL, Catalogue no SM065F, Acris Antibodies GmbH,Herford, Germany) or anti-7/4-FITC (2 μg/mL, Catalogue no CL050F, AcrisAntibodies GmbH, Herford, Germany) for 30 min at 30° C., and thencounterstained with propidium iodide (2 μg/mL) to allow exclusion ofnecrotic cells. The cells were washed with PBS, and transferred to FACStubes. Samples was set in a flow cytometer (ALTRA II; Beckman CoulterJapan, Tokyo, Japan), and a single-parameter FL2 [PMT2] (FITC) histogramwith log x-axis was plotted to illustrate relative intensity of FITC,Data files were stored for subsequent analysis using Kaluza analysissoftware (ver. 1.2) to calculate the proportion of each cell type.

Cigarette Smoke Model

A/J mice (males, 5 weeks old) were exposed to cigarette smoke (4%cigarette smoke, diluted with compressed air) for 30 min/day for 11 daysusing a Tobacco Smoke Inhalation Experiment System for small animals(Model SIS-CS; Sibata Scientific Technology, Tokyo, Japan). Testsubstances were administered once daily for 3 days after the finalcigarette smoke exposure (intra-nasal dose comprising 35 μL of solutionin 50% DMSO/isotonic saline).

At 12 hr after administration of the last dose, animals wereanesthetized, the trachea was cannulated and bronchial alveolar lavage(BAL) was carried out by the instillation into and then withdawal fromthe lungs, of PBS (100 mL/kg). Total cell numbers in the BALF sampleswere measured using a haemocytometer under a phase-contrast microscope.The proportions of alveolar macrophages and neutrophils were determinedby FACS analysis using anti-mouse MOMA2-FITC (macrophage) or anti-mouse7/4 (neutrophil).

Cells were suspended in PBS and were incubated with anti-MOMA2-FITC (2μg/mL, Catalogue no SM065F, Acris Antibodies GmbH, Herford, Germany) oranti-7/4-FITC (2 μg/mL, Catalogue no CL050F, Acris Antibodies GmbH,Herford, Germany) for 30 min at 30° C., and also counterstained withpropidium iodide (2 μg/mL) to allow exclusion of necrotic cells. Thecells were washed with PBS, and transferred to FACS tubes. Samples wasset in a flow cytometer (ALTRA II; Beckman Coulter Japan, Tokyo, Japan),and a single-parameter FL2 [PMT2] (FITC) histogram with log x-axis wasplotted to illustrate relative intensity of FITC.

Data files are stored for subsequent analysis using Kaluza analysissoftware (ver. 1.2) to calculate the proportion of each cell type. Thelevels of CXCL1(KC), MCP1, TNFα, IL-17 or osteopontin in BALF weredetermined using Quentikine® mouse KC, MCP1, TNFα, IL-17 or osteopontinELISA kit (R&D systems, Inc., Minneapolis, Minn., USA). The presence ofmalondialdehyde was measured using OxiSelect® TBARS Assay Kit (MDAQuantitation; Cell Biolabs Inc, San Diego, Calif., USA).

Summary of In Vitro and In Vivo Screening Results

The in vitro profile of the compound of formula (I), as determined usingthe methods described above is presented below (Tables 1, 2 and 3). Thecompound of the present invention demonstrates potent inhibition of bothPI3K δ and γ isoforms, and shows no inhibitory activity versus PI3K αand only low inhibitory activity versus PI3K β in enzyme assays (Table1).

These effects translate into potent inhibition of Akt phosphorylationinduced by the stimulation of cells with either hydrogen peroxide orMCP-1, No effects on cell viability, resulting from incubation with thecompound of formula (I), were detected (Table 2). Furthermore treatmentof cells with the compound of formula (I) disclosed herein was found toinhibit production of ROS from U937 cells and of cytokines fromcytostim-challenged PBMCs (Table 3).

It is notable that the likely metabolic product of the compound offormula (I), namely corresponding alcohol, compound (Ia), is asignificantly less active inhibitor of both PI3K δ and γ isoforms thanthe compound of formula (I) (Table 2). Consequently the compound offormula (Ia), is a significantly less active inhibitor of ROS productionfrom U937 cells and of cytokines from cytostim-challenged PBMCs (Table3) than the compound of formula (I).

TABLE 1 Comparison of the PI3K isoform inhibitory activities of priorart compounds with Compound (I). Compound A

Example 50

IC50 Values for PI3K inhibition Test at isozyme indicated (nM) CompoundsPI3K α PI3K β PI3K δ PI3K γ Compound (I) >13900 4890  2.5  28 CompoundA¹    193 NT 12    25 Example 50²    653 NT  5.7 120 ¹Prior art compounddisclosed in WO 2012/052753; ²Prior art compound disclosed in WO2011/048111; NT not tested;

TABLE 2 The inhibitory activity of compounds (I) and (Ia) on PI3K enzymeisoforms; the inhibition of inducd phosphylation of Akt in cells and oncell viability. Cellular Activity PI3 Kinase Inhibition REC₅₀ values^(a)in d- Cell Viability IC₅₀ value U937 cells (nM) MTT Assay^(b) Test atstate isozyme (nM) H₂O₂ MCP-1 in d-U937 cells Cmpd δ γ α β stimulusstimulus at 4 hr at 24 hr (I) 2.5 28 >13900  4890 7.4 16.5 −ve −ve (Ia)17 577 >14200 >14200 ND ND −ve −ve ^(a)for the inhibition of induced Aktphosphorylation; ^(b)−ve indicates a value of <30% inhibition at 10μg/mL; ND: not done

TABLE 3 The effects of the compounds of formula (I) and (Ia) on ROSproduction from U937 cells and cytokine release from PBMCs REC₅₀ ¹ orIC₅₀ ² value (nM) Cellular system Compound (I) Compound (Ia)Zymosan-induced ROS^(a) production 11.8 143 in IFNγ-primed U937 cells¹Cytostim-induced IL-4 in PBMCs² <1.4 674 Cytostim-induced IL-5 in PBMCs²<1.4 14.5 Cytostim-induced IL-13 in PBMCs² <1.4 6.1 Cytostim-inducedIFNγ in PBMCs² 13.8 60.8 THP1 cell chemotaxis to MCP1¹ 33.9 >14200TNFα-induced CXCL8 in neutrophils  2.2 ND from COPD patients¹ a) ROS:reactive oxygen species.

The effects of treatment with compound (I) on LPS-induced airwayneutrophilia in mice and rats are reported in Tables 4 and 5,respectively. Treatment was found to produce a dose-dependent inhibitionof LPS-induced neutrophilia in both species. Furthermore, the inhibitoryeffects of treatment on cell accumulation were found to demonstrate along duration of action.

TABLE 4 The Effects of Treatment with Compound (I) on LPS-induced airwayneutrophilia in mice. Neutrophil numbers in BALF (x10⁵/mL, mean ± SEM)Compound (I) at pre-dose time indicated (% inhibition) (mg/mL) 2 hr 8 hr12 h Vehicle 17.1 ± 2.5    — —  0.05 13.8 ± 2.5 (19)  — — 0.2 8.0 ± 1.4(53) 9.4 ± 2.0 (45) 13.1 ± 2.5 (23) 1.0 5.5 ± 0.9 (68) — — N = 8 animalsper group

TABLE 5 The Effects of Treatment with Compound (I) on LPS-Induced AirwayNeutrophilia in Rats. Neutrophil numbers in BALF (x10⁵/mL, mean ± SEM)Compound (I) at pre-dose time indicated (% inhibition) (mg/mL) 2 hr 8 hr12 h Vehicle 15.1 ± 2.6   — —  0.05   13.2 ± 2.3 (9) — — 0.2    6.3 ±1.6 (58) 10.1 ± 1.8 (33) 13.6 ± 2.7 (10) 1.0    4.1 ± 0.7 (73) — — N = 8animals per group

The effects of treatment with compound (I) on allergen challenge-inducedairway eosinophilia and neutrophilia in mice are reported in Table 6.Treatment of mice with the compound disclosed herein was found toproduce a dose-dependent inhibition of both eosinophil and neutrophilaccumulation in bronchoalveolar lavage following allergen challenge.

TABLE 6 The effects of treatment with Compound (I) on ovalbumin-inducedairway eosinophilia and neutrophilia in ovalubumin sensitized mice. Cellnumbers in BALF Compound (I) (x10⁴/mL, mean ± SEM) and (% inhibition)(mg/mL) Eosinophils Neutrophils Vehicle 24.7 ± 3.1   9.8 ± 0.6  0.05   19.7 ± 3.2 (20)    8.0 ± 0.4 (18) 0.2    3.8 ± 0.9 (85)    2.9 ± 0.5(70) 1    2.1 ± 0.4 (91)    2.0 ± 0.3 (80) N = 8 animals per group

The effect of treatment with compound (I) or with compound A onmacrophage and neutrophil accumulation in BALF following exposure ofmice to poly-I:C was also investigated. In this direct comparison,treatment with either compound (I) or compound A was found to produce adose-dependent inhibition of poly-I:C-induced macrophage and neutrophilaccumulation into BALF (Table 7). It is notable that compound (I) showssignificantly greater potency than compound A and this data isrepresented graphically for neutrophils (FIG. 1)

TABLE 7 The effects of treatment with Compound (I) or Compound A onpoly-I:C-induced cell accumulation in mice airways. Treatment and doseof Cell numbers in BALF^(a) (x10⁴/mL) Compound (I) or Compound A and (%inhibition) (mg/mL) Macrophages Neutrophils Vehicle 3.6 ± 0.2  1.4 ±0.1  Vehicle + Poly I:C 19.0 ± 0.5   11.6 ± 0.2   Compound (I) (0.002) +Poly I:C    11.5 ± 0.2 (49)    7.3 ± 0.3 (43) Compound (I) (0.02) + PolyI:C    8.3 ± 0.3 (69)    5.0 ± 0.2 (65) Compound (I) (0.2) + Poly I:C   6.8 ± 0.3 (79)    3.8 ± 0.2 (77) Compound A (0.02) + Poly I:C    14.3± 0.3 (31)    8.9 ± 0.2 (26) Compound A (0.2) + Poly I:C    11.0 ± 0.4(52)    7.3 ± 0.3 (42) Compound A (2) + Poly I:C    7.8 ± 0.2 (73)   4.8 ± 0.2 (67) a) The data for cell numbers are shown as the mean ±SEM; N = 5 animals per group

TABLE 8 The effect of treatment with Compound (I) ± fluticasonepropionate on cigarette smoke (CS)-induced cell accumulation in murineBALF. Cell numbers in BAL^(b) (x10⁴/mL) Treatment and dose of and (%inhibition) Compound (I) (mg/mL) Macrophages Neutrophils Air + Vehicle4.5 ± 0.3  1.5 ± 0.2  CS + Vehicle 17.9 ± 0.6   11.1 ± 0.5   CS +Compound (I) (0.02)    13.6 ± 0.2 (32)    8.6 ± 0.2 (26) CS + Compound(I) (0.2)    9.5 ± 0.2 (63)    6.0 ± 0.1 (54) CS + Compound (I) (2)   7.4 ± 0.3 (79)    4.6 ± 0.1 (68) CS + Compound (I) (0.02) + FP^(a)   13.6 ± 0.4 (32)    8.7 ± 0.2 (26) CS + Compound (I) (0.2) + FP^(a)   9.4 ± 0.3 (63)    5.8 ± 0.3 (55) CS + Compound (I) (2) + FP^(a)   7.0 ± 0.3 (81)    4.3 ± 0.2 (71) N = 5 animals per group; a) FP =fluticasone propionate dosed at 50 μg/mL; b) The data for cell numbersare shown as the mean ± SEM,

The effects of treatment with compound (I) on macrophage and neutrophilaccumulation in BALF following exposure to cigarette smoke wasdetermined (Table 8). The cigarette smoke model used for this study isreported to be a corticosteroid refractory system [To, Y. et al., Am. J.Respir. Crit. Care Med., 2010, 182:897-904; Medicherla, S. et al., J.Pharmacol. Exp. Ther. 2008, 324:921-9] and the data reveal thatdexamethasone (0.3-10 mg/kg, p.o.) was, as anticipated, inactive. Theeffects of treatment with compound (I) on BALF neutrophils and onactivated alveolar macrophage numbers demonstrate that it possessesanti-inflammatory activity when administered as a monotherapy. Moreover,when compound (I) was co-administered with fluticasone propionate, at adose which lacks any significant effect as monotherapy, a markedenhancement of anti-inflammatory activity was detected. In acontemporaneous study, the effects of treatment, with compound A, ofmice exposed to cigarette-smoke were evaluated. A comparison of thesedata with those presented above for compound (I), demonstrates thatcompound (I) is a more potent inhibitor of cigarette-smoke induced cellaccumulation in murine BALF than is compound A (FIG. 2).

Tobacco smoke exposure also increased the concentrations of theinflammatory biomarkers CXCL1, MCP1, TNFα, IL17, osteopontin andmalondialdehyde in bronchoalveolar lavage fluid. Treatment with compound(I) decreased the concentrations of each of the biomarkers in adose-dependent manner. Furthermore, treatment with both compound (I) andfluticasone propionate in combination showed greater decreases inbiomarker concentrations than was achieved with treatment with compound(I) alone (Table 9).

TABLE 9 The effect of treatment with Compound (I) ± fluticasonepropionate on biomarkers in murine BALF. Concentration of biomarker inBALF Inhibition¹ (%) against treatment Compound (I) Compound (I) (pg/mL)(mg/mL) (mg/mL) + FP² Biomarker Air Tobacco 0.02 0.2 2 0.002 0.02 0.2CXCL1 8.4 ± 0.1  18 ± 0.3 32 52 71 32 58 77 MCP-1 2.3 ± 0.2 7.2 ± 0.1 2845 78 29 53 73 TNF α  1.5 ± 0.04 3.6 ± 0.1 22 38 62 22 39 64 IL-17 1.2 ±0.1 2.7 ± 0.1 27 41 60 26 51 67 Osteopontin  11 ± 0.3  23 ± 0.4 22 44 6321 45 62 MDA  0.3 ± 0.02³  1.6 ± 0.04³ 25 42 64 29 43 64 N = 5 animalsper group; ¹Percentage inhibition with respect to tobacco, ²fluticasonepropionate (0.5 mg/mL); ³These data are are μM values; are smoke controlafter subtracted air control values

In summary, the compound of the invention is a potent inhibitor of bothPI3K δ and γ isoforms. The in vitro profile translates into a broadanti-inflammatory phenotype in vivo. In this setting, the inhibitoryeffects of the compound disclosed herein versus Poly I:C-induced cellaccumulation in the airways is notable. It is also particularly strikingthat, unlike selective inhibitors of PI3K δ, treatment with compound (I)disclosed herein alone results in marked inhibition of cigarette-smokeinduced airways inflammation and that these effects occur at lower doseswhen it is co-administered with a corticosteroid, fluticasonepropionate, under conditions where treatment with the corticosteroidalone is without effect.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer, step, group of integers or group of steps but notto the exclusion of any other integer, step, group of integers or groupof steps.

All patents and patent applications referred to herein are incorporatedby reference in their entirety.

1. A compound of formula (I)

or a pharmaceutically acceptable salt thereof, including allstereoisomers, tautomers and isotopic derivatives thereof.
 2. Apharmaceutical composition comprising a compound according to claim 1,in combination with one or more pharmaceutically acceptable diluents orcarriers.
 3. A pharmaceutical composition according to claim 2 furthercomprising a second or further active ingredient selected fromcorticosteroids, beta agonists, xanthenes, musacarinic antagonists andp38 MAP kinase inhibitors.
 4. A combination product comprising: (A) acompound of formula (I) according to claim 1; and (B) a further activeingredient selected from corticosteroids, beta agonists, xanthenes,musacarinic antagonists and p38 MAP kinase inhibitors, wherein each ofcomponents (A) and (B) is formulated in admixture with apharmaceutically-acceptable diluent or carrier.
 5. A compound of formula(I) according to claim 1 for use as a medicament.
 6. A compound offormula (I) according to claim 1 for use as a medicament to beadministered in combination with one or more further active ingredientsselected from corticosteroids, beta agonists, xanthenes, musacarinicantagonists and p38 MAP kinase inhibitors.
 7. A compound of formula (I)according to claim 1 for use in the treatment or prevention of acondition selected from the group consisting of: COPD (including chronicbronchitis and emphysema), asthma, paediatric asthma, cystic fibrosis,sarcoidosis, idiopathic pulmonary fibrosis, allergic rhinitis, rhinitis,sinusitis, and viral induced exacerbations of any one of the same,respiratory virus infection (including the complications thereof),allergic conjunctivitis, conjunctivitis, allergic dermatitis, contactdermatitis, psoriasis, ulcerative colitis, inflamed joints secondary torheumatoid arthritis or osteoarthritis, rheumatoid arthritis,pancreatitis, cachexia, inhibition of the growth and metastasis oftumours including non-small cell lung carcinoma, breast carcinoma,gastric carcinoma, colorectal carcinomas and malignant melanoma.
 8. Useof a compound of formula (I) according to claim 1 for the manufacture ofa medicament for the treatment or prevention of a condition selectedfrom the group consisting from: COPD (including chronic bronchitis andemphysema), asthma, paediatric asthma, cystic fibrosis, sarcoidosis,idiopathic pulmonary fibrosis, allergic rhinitis, rhinitis, sinusitisand virally-induced exacerbations of any one of the same, respiratoryvirus infection (including the complications thereof), allergicconjunctivitis, conjunctivitis, allergic dermatitis, contact dermatitis,psoriasis, ulcerative colitis, inflamed joints secondary to rheumatoidarthritis or osteoarthritis, rheumatoid arthritis, pancreatitis,cachexia, inhibition of the growth and metastasis of tumours includingnon-small cell lung carcinoma, breast carcinoma, gastric carcinoma,colorectal carcinomas and malignant melanoma.
 9. A method of treatmentof a condition selected from the group consisting from: COPD (includingchronic bronchitis and emphysema), asthma, paediatric asthma, cysticfibrosis, sarcoidosis, idiopathic pulmonary fibrosis, allergic rhinitis,rhinitis, sinusitis, and virally-induced exacerbations of any one of thesame, respiratory virus infection (including the complications thereof),allergic conjunctivitis, conjunctivitis, allergic dermatitis, contactdermatitis, psoriasis, ulcerative colitis, inflamed joints secondary torheumatoid arthritis or osteoarthritis, rheumatoid arthritis,pancreatitis, cachexia, inhibition of the growth and metastasis oftumours including non-small cell lung carcinoma, breast carcinoma,gastric carcinoma, colorectal carcinomas and malignant melanoma whichcomprises administering to a subject in need thereof an effective amountof a compound of formula (I) according to claim
 1. 10. An intermediateof formula (II):

wherein LG₁ represents a leaving group or a protected derivate thereof.11. A process for preparing a compound of formula (I) comprisingreacting a compound of formula (II):

or a protected derivative thereof, wherein LG₁ represents a leavinggroup, with a fragment:

under conditions appropriate to provide compound of formula (I) or aprotected derivative thereof and, if necessary, deprotecting theprotected compound to yield a compound of formula (I).
 12. A compound offormula (Ia)

or a pharmaceutically acceptable salt thereof, including allstereoisomers, tautomers and isotopic derivatives thereof